Novel Triterpene-Glycosides As Sweeteners Or Sweetener Enhancer
(19) T ZZ¥ Z_ _T
(11) EP 3 501 296 A1
(12) EUROPEAN PATENT APPLICATION
(43) Date of publication: (51) Int Cl.: 26.06.2019 Bulletin 2019/26 A23L 27/30 (2016.01) C07J 17/00 (2006.01)
(21) Application number: 17210082.8
(22) Date of filing: 22.12.2017
(84) Designated Contracting States: • Kluge, Grit AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 14959 Trebbin (DE) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • Jakupovic, Sven PL PT RO RS SE SI SK SM TR 13591 Berlin (DE) Designated Extension States: •Hetterling,Gregor BA ME 12057 Berlin (DE) Designated Validation States: • Tsichrintzi, Fotini MA MD TN 10707 Berlin (DE)
(71) Applicant: Analyticon Discovery GmbH (74) Representative: Fabry, Bernd 14473 Potsdam (DE) IP2 Patentanwalts GmbH Schlossstrasse 523 (72) Inventors: 41238 Mönchengladbach (DE) • Dr. Siems, Karsten 14552 Michendorf (DE)
(54) NOVEL TRITERPENE-GLYCOSIDES AS SWEETENERS OR SWEETENER ENHANCER
(57) Suggested are triiterpene-glycoside com- pounds, which are obtainable by the extraction of Gynos- temma pentaphyllum which are useful as a sweetener or sweet taste enhancer in preparations and compositions, especially oral edible compositions. EP 3 501 296 A1
Printed by Jouve, 75001 PARIS (FR) EP 3 501 296 A1
Description
FIELD OF INVENTION
5 [0001] The invention relates to novel triterpene-glycoside compounds, which are obtainable by the extraction of Gy- nostemma pentaphyllum and its physiologically acceptable salts which are useful as a sweetener or sweetener enhancer in preparations and compositions, especially oral edible compositions.
STATE OF THE ART 10 [0002] Sweetness is one of the primary taste and cravings of both animals and humans. The universal use of naturally occurring and synthetic sweeteners to satisfy this natural craving has not been met without its accompanying physiological disadvantages, e.g. obesity, nutritional imbalance and dental decay. To overcome these unwanted disadvantages con- siderable research efforts and expenditures have been made to develop alternative compounds, e.g. as substitute for 15 the naturally occurring sweeteners or synthetic sweeteners which have no food value and are free of caloric input. While these artificial sweeteners enjoyed a wide use, and fulfilled the requirements of a sweet taste with no food value, and could be used without providing calories or damaging teeth, they were frequently found to possess inherent disadvantages that prevented their use for their intended purpose, e.g. because of their toxicity (p-ethoxyphenylurea) or chromosome damage and bladder trouble (sodium cyclamate). Thus, these sweeteners could not be safely recommended for use as 20 a sweetener and are apparently unacceptable for consumption. Saccharin compounds are also commonly used as artificial sweeteners, since cyclamates have been come under governmental restrictions. Although saccharin compounds possess sweetness characteristics, they are undesirable as the sole sweetening agent in most food and beverage compositions because of the lingering bitter aftertaste perceived by most users. While saccharin and the cyclamates have been in common use as artificial sweetening agents for a number of years, there has been more recently discovered 25 a series of new artificial sweeteners. [0003] For example US 3,087,821 (GENTILI ET AL) teaches the use of various dihydrochalcones having sugar sub- stituents (dihydrochalcones glycosides) as sweetening agents. All sweet dihydrochalkones have a licorice like aftertaste and linger in the mouth for some time. [0004] Siraitia grosvenori (Luo han guo), a member of the Cucurbitaceae family, is a plant native to some regions of 30 southern Asia and China. The sweet taste of fruits of luo han guo mainly comes from triterpene glycosides generally known as mogrosides. There are a number of mogrosides identified in luo han guo but generally mogroside V (CAS No: 88901-36-4)has the highest concentrationcompared toothers (Table1). Mogrol glycosides have the same core molecule- mogrol or oxo-mogrol and differ from each other by number and type of glycosidic residues bonded to mogrol or oxo- mogrol molecules [see US 2012/0059071, Kasai et al. AGRIC BIOL CHEM (1989), p 3347-3349; Matsumoto et. al. 35 CHEM PHARM BULL (1990) p 2030-2032.] Several mogrosides taste very sweet, often >100x sweeter than sucrose (EP 3099186 A2 , ANALYTICON DISCOVERY), including the major triterpene glycoside of Siraitia grosvenori, mogroside V, and its isomer iso-mogroside V (US 20110027413), but all of them have a certain bitter aftertaste. [0005] Leaves of Stevia rebaudiana are well known for its sweet taste due to its content of sweet diterpene glycosides. One of the major sweet compounds from stevia, Rebaudioside A, is approved as natural sweetener in US (since 2008) 40 und EU (since 2011). Apart from its sweet taste, all sweeteners from stevia have a slower onset and longer duration than that of sugar, and a bitter or licorice-like aftertaste at high concentrations [Lemus-Mondaca et al. FOOD CHEM 132 (2012) p 1121-1132]. [0006] Another example is EP 2529633 A1 (SYMRISE), which relates to triterpenes and triterpene glycosides and/or physiologically acceptable salts thereof. The triterpenes are, preferably naturally occurring triterpenes and triterpene 45 glycosidesfrom Mycetiabalansae, for generating asweet impressionin an orally consumableformulation or forreinforcing the sweet impression of an orally consumable formulation. These triterpene-glycosides differ in structure from the trit- erpenes claimed in the present invention. [0007] Gynostemma pentaphyllum is traditionally used as a natural sweetening agent in China. At least 85 triterpene glycosides were isolated from G. pentaphyllum, but only Gypenoside XX is known for its sweet taste (KINGHORN et 50 al., Terpenoid Glycoside Sweeteners in Natural Occurring Glycosides: Chemistry, Distribution and Biological Properties. John Wiley and Sons. Chichester, UK, 1999, p. 399-429.) [0008] Accordingly, it is a primary object of the present invention to provide novel sweetener compounds and its physiologically acceptable salts, which have a positive sweet benefit in food and oral compositions. In particular, the object was to provide sweetener compounds which are capable to provide sweetness to consumable compositions in 55 a way, that the balance between the degree of sweetness and the amount which has to be administered to obtain a sweet effect is comparable low, to overcome the aforesaid disadvantages associated with the prior art sweetener. It is another object of the present invention to provide sweetener compounds without astringent or bitter-taste aftertaste. The sweetener compounds to be specified should be toxicologically safe, effective already at relatively low concentrations,
2 EP 3 501 296 A1
well tolerated by the digestion, stable (in particular in normal cosmetic and/or pharmaceutical formulations), and easy to formulate and economical to produce.
DESCRIPTION OF THE INVENTION 5 [0009] Object of the present invention is a sweetener composition comprising or consisting of at least one compound of general formula (I)
10
15
20
25
30 wherein
R1, R1a and R1b are independent from each other and represent OH, OGlucose, or OXylose, or OGalaktose, or OAra- binose
R2 is one of the following structures 35
40
45
50 with
A1, A2 and A3 are independent from each other and represent H, OH, OOH, or OMe and
B1 and B2 are independent from each other and represent H, OH, OOH, OMe, or O 55 and the dotted line means a single or a double bond. [0010] In the course of extensive studies on sweeteners, the present inventors succeeded in the isolation of novel sweetener compounds of formula (I) and found that these sweetener compounds of formula (I) show astonishingly good
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and strong sweetness when compared with that of sucrose.
PREFERRED COMPOUNDS
5 [0011] Of particular interest are the compounds of Formula (I) identified as:
Compound A (NP-019652)
[0012] 10
15
20
25
Compound B (NP-019654) 30 [0013]
35
40
45
50
Compound C (NP-019869) 55 [0014]
4 EP 3 501 296 A1
5
10
15
20
Compound D (NP-019870)
25 [0015]
30
35
40
45
Compound E (NP-019871)
50 [0016]
55
5 EP 3 501 296 A1
5
10
15
20
Compound F (NP-022163)
[0017] 25
30
35
40
45
Compound G (NP-022167)
[0018] 50
55
6 EP 3 501 296 A1
5
10
15
20
Compound H (NP-021249)
25 [0019]
30
35
40
45
Compound I (NP-021250)
50 [0020]
55
7 EP 3 501 296 A1
5
10
15
20
25 Compound J (NP-021251)
[0021]
30
35
40
45
50 Compound K (NP-021252)
[0022]
55
8 EP 3 501 296 A1
5
10
15
20
Compound L (NP-021253)
25 [0023]
30
35
40
45
EXTRACTS 50 [0024] Another object of the present invention refers to an extract comprising one or more of a compound of formula (I) obtainable by aqueous and/or alcoholic extraction of the plant Gynostemma pentaphyllum. Particularly preferred are extracts prepared from the leaves of G. pentaphyllum.
55 EXTRACTION PROCESS
[0025] The extracts according to the present invention may be prepared by methods known per se, i.e. for example by aqueous, alcoholic or aqueous/alcoholic extraction of the plants or parts thereof. Suitable extraction processes are
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any conventional extraction processes, such as maceration, re-maceration, digestion, agitation maceration, vortex ex- traction, ultrasonic extraction, counter current extraction, percolation, re-percolation, evacolation (extraction under re- duced pressure), diacolation and solid/liquid extraction under continuous reflux. Percolation is advantageous for industrial use. 5 [0026] The plant materials that are useful for obtaining the abstracts may include parts of the whole plant selected from the group consisting of blossoms, fruits, buds, roots, seeds and/or leaves or the whole plant itself. Leaves, however, are preferably used as the starting material and may be mechanically size-reduced before the extraction process. Any size reduction methods known to the expert, for example freeze grinding, may be used. Preferred solvents for the extraction process are organic solvents, water (preferably hot water with a temperature above 80 °C and more particularly 10 above 95 °C or mixtures of organic solvents and water, more particularly low molecular weight alcohols with more or less high water contents. Extraction with methanol, ethanol and water-containing mixtures thereof is particularly preferred. The extraction process is generally carried out at about 20 to about 100 °C and preferably at about 30 to about 70 °C. In one preferred embodiment, the extraction process is carried out in an inert gas atmosphere to avoid oxidation of the ingredients of the extract. This is particularly important where extraction is carried out at temperatures above 40 °C. The 15 extraction times are selected by the expert in dependence upon the starting material, the extraction process, the extraction temperature and the ratio of solvent to raw material, etc. After the extraction process, the crude extracts obtained may optionally be subjected to other typical steps, such as for example purification, concentration and/or decoloration. If desired, the extracts thus prepared may be subjected, for example, to the selective removal of individual unwanted ingredients. The extraction process may be carried out to any degree, but is usually continued to exhaustion. Typical 20 yields (=extract dry matter, based on the quantity of raw material used) in the extraction of the starting materials are of the order of about 5 to about 35, %, preferably about 10 to about 30 and more preferably about 15 to about 25 % b.w. - calculated on the starting materials. Particular preferred is a process for obtaining an extract of Gynostemma penta- phyllum rich in compounds according to formula (I) encompassing the steps of:
25 (a)Providing asuspension of leaves of Gynostemmapentaphyllum in water, a C 1-C4 aliphatic alcohol or their mixtures
(b) Subjecting said suspension to extraction using water, a C 1-C4 aliphatic alcohol or their mixtures at temperatures of about 30 to about 70 °C, and optionally
30 (c) Separating off the solvent.
[0027] It should be noted that the content in the extract of each of the compounds (A) to (L) differs from batch to batch depending on the used raw material of leaves of Gynostemma pentaphyllum.
35 COMPOSITIONS
[0028] The preparation of the present invention comprising at least one sweetener compound of formula (I) and can be used to impart a desirable sweetness and/ or flavor to a variety of oral and food compositions and pharmaceutical compositions, such as beverages, edible foodstuff, dentifrices, lipsticks and the like, which may or may not be ingestible, 40 with or without the use of other flavorants and sweeteners. The present invention also relates to a variety of oral and food compositions and the like embodying at least one compound of formula (I) as sweetener and/ or flavoring agent. [0029] The sweetener of this invention finds application in the wide range of edible substances generally, primarily in food compositions such as candies, confections and processed foods, and beverages such as beer and soft drinks. It is also well suited for imparting a sweet flavor to other edible substances such as medicines, toothpaste, adhesives for 45 stamps and envelopes, animal feeds and baits and the like. These examples are given solely for illustration and it is not wished to limit the scope of this invention to sweetening any particular type or types of edible materials. As a general rule, the present sweetener may be used in any application where a sweet taste is desired. The present sweetener may be used alone or in combination with other sweeteners, nutritive or nonnutritive. Also, if desired, binders or diluents may be added to the sweetener. This is not usually necessary, however, as the sweetener is a solid having excellent handling 50 properties. This makes mixing the sweetener with an edible substance a simple conventional operation. The sweetener may be mixed with the edible substance as a solid or as a solution, if desired. [0030] The inventions further refers to the use of a sweetener compound of formula (I) to sweet or enhance the sweeting effect in compositions or preparations which are administered to an individual in an effective amount sufficient to produce the desired degree of sweetness. 55 [0031] Thus the invention further relates to a method for providing a sweetening effect and/or an enhanced sweetening effect in compositions, comprising administering to an individual a sweetener compound of formula (I) in an effective amount sufficient to produce the desired degree of sweetness. [0032] The effective amount is preferably from 1 ppm to 2000 ppm, based on the total weight of the composition and
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the total sum of all compound of formula (I). [0033] The food, oral and pharmaceutical compositions will be further described in detailed.
FOOD COMPOSITIONS 5 [0034] Another object of the present invention refers to a food composition, comprising the sweetener composition or the extract as disclosed above. [0035] Food compositions according to the invention are any preparations or compositions which are suitable for consumption and are used for nutrition or enjoyment purposes, and are generally products which are intended to be 10 introduced into the human or animal oral cavity, to remain there for a certain time and then either be eaten (e.g. ready- to-eat foodstuffs or feeds, see also herein below) or removed from the oral cavity again (e.g. chewing gums). Such products include any substances or products which in the processed, partially processed or unprocessed state are to be ingested by humans or animals. They also include substances which are added to orally consumable products during their manufacture, preparation or treatment and which are intended to be introduced into the human or animal oral cavity. 15 [0036] The food compositions according to the invention also include substances which in the unchanged, treated or prepared state are to be swallowed by a human or animal and then digested; in this respect, the orally consumable products according to the invention also include casings, coatings or other encapsulations which are to be swallowed at the same time or which may be expected to be swallowed. The expression "orally consumable product" covers ready- to-eat foodstuffs and feeds, that is to say foodstuffs or feeds that are already complete in terms of the substances that 20 are important for the taste. The expressions "ready-to-eat foodstuff" and "ready-to-eat feed" also include drinks as well as solid or semi-solid ready-to-eat foodstuffs or feeds. Examples which may be mentioned are frozen products, which must be thawed and heated to eating temperature before they are eaten. Products such as yoghurt or ice-cream as well as chewing gums or hard caramels are also included among the ready-to-eat foodstuffs or feeds. [0037] Preferred food compositions according to the invention also include "semi-finished products". Within the context 25 of the present text, a semi-finished product is to be understood as being an orally consumable product which, because of a very high content of flavorings and taste-imparting substances, is unsuitable for use as a ready-to-eat orally con- sumable product (in particular foodstuff or feed). Only by mixing with at least one further constituent (e.g. by reducing the concentration of the flavorings and taste-imparting substances in question) and optionally further process steps (e.g. heating, freezing) is the semi-finished product converted into a ready-to-eat orally consumable product (in particular 30 foodstuff or feed). Examples of semi-finished products which may be mentioned here are [0038] Food composition according to the invention preferably comprises one or more preparations for nutrition or enjoyment purposes. These include in particular (reduced-calorie) baked goods (e.g. bread, dry biscuits, cakes, other baked articles), confectionery (e.g. chocolates, chocolate bars, other products in bar form, fruit gums, dragees, hard and soft caramels, chewing gum), non-alcoholic drinks (e.g. cocoa, coffee, green tea, black tea, (green, black) tea drinks 35 enriched with (green, black) tea extracts, rooibos tea, other herbal teas, fruit-containing soft drinks, isotonic drinks, refreshing drinks, nectars, fruit and vegetable juices, fruit or vegetable juice preparations), instant drinks (e.g. instant cocoa drinks, instant tea drinks, instant coffee drinks), meat products (e.g. ham, fresh sausage or raw sausage prepa- rations, spiced or marinated fresh or salt meat products), eggs or egg products (dried egg, egg white, egg yolk), cereal products (e.g. breakfast cereals, muesli bars, precooked ready-to-eat rice products), dairy products (e.g. full-fat or 40 reduced-fat or fat-free milk drinks, rice pudding, yoghurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, partially or completely hydrolysed milk-protein-containing products), products made from soy protein or other soybean fractions (e.g. soy milk and products produced therefrom, drinks containing isolated or enzymatically treated soy protein, drinks containing soy flour, preparations containing soy lecithin, fermented products such as tofu or tempeh or products produced therefrom and mixtures with fruit preparations and optionally flavors), fruit 45 preparations (e.g. jams, sorbets, fruit sauces, fruit fillings), vegetable preparations (e.g. ketchup, sauces, dried vegeta- bles, frozen vegetables, precooked vegetables, boiled-down vegetables), snacks (e.g. baked or fried potato crisps or potato dough products, maize- or groundnut-based extrudates), fat- and oil-based products or emulsions thereof (e.g. mayonnaise, remoulade, dressings, in each case full-fat or reduced-fat), other ready-made dishes and soups (e.g. dried soups, instant soups, precooked soups), spices, spice mixtures and in particular seasonings which are used, for example, 50 in the snacks field, sweetener preparations, tablets or sachets, other preparations for sweetening or whitening drinks or other foods. The preparations within the scope of the invention can also be used in the form of semi-finished products for the production of further preparations for nutrition or enjoyment purposes. The preparations within the scope of the invention can also be in the form of capsules, tablets (uncoated and coated tablets, e.g. enteric coatings), dragees, granules, pellets, solids mixtures, dispersions in liquid phases, in the form of emulsions, in the form of powders, in the 55 form of solutions, in the form of pastes, or in the form of other preparations which can be swallowed or chewed, and in the form of food supplements. [0039] The preparations can also be in the form of capsules, tablets (uncoated and coated tablets, e.g. enteric coatings), dragees, granules, pellets, solids mixtures, dispersions in liquid phases, in the form of emulsions, in the form of powders,
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in the form of solutions, in the form of pastes, or in the form of other preparations which can be swallowed or chewed, for example in the form of food supplements. [0040] The semi-finished products are generally used for the production of ready-to-use or ready-to-eat preparations for nutrition or enjoyment purposes. 5 [0041] Further constituents of a ready-to-eat preparation or semi-finished product for nutrition or enjoyment purposes can be conventional base substances, auxiliary substances and additives for foods or enjoyment foods, for example water, mixtures of fresh or processed, vegetable or animal base or raw substances (e.g. raw, roast, dried, fermented, smoked and/or boiled meat, bone, cartilage, fish, vegetables, herbs, nuts, vegetable juices, vegetable pastes or mixtures thereof), digestible or non-digestible carbohydrates (e.g. sucrose, maltose, fructose, glucose, dextrins, amylose, amy- 10 lopectin, inulin, xylans, cellulose, tagatose), sugar alcohols (e.g. Sorbitol, erythritol), natural or hardened fats (e.g. tall ow, lard, palm fat, cocoa fat, hardened vegetable fat), oils (e.g. sunflower oil, groundnut oil, maize germ oil, olive oil, fish oi l, soya oil, sesame oil), fatty acids or their salts (e.g. potassium stearate), proteinogenic or non-proteinogenic amino acids and related compounds (e.g. γ-aminobutyric acid, taurine), peptides (e.g. glutathione), natural or processed proteins (e.g. gelatin), enzymes (e.g. peptidases), nucleic acids, nucleotides, taste correctors for unpleasant taste impressions, 15 further taste modulators for further, generally not unpleasant taste impressions, other taste-modulating substances (e.g. inositol phosphate, nucleotides such as guanosine monophosphate, adenosine monophosphate or other substances such as sodium glutamate or 2-phenoxypropionic acid), emulsifiers (e.g. lecithins, diacylglycerols, gum arabic), stabilisers (e.g. carrageenan, alginate), preservatives (e.g. benzoic acid and its salts, sorbic acid and its salts), antioxidants (e.g. tocopherol, ascorbic acid), chelators (e.g. citric acid), organic or inorganic acidifying agents (e.g. acetic acid, phosphoric 20 acid), additional bitter substances (e.g. quinine, caffeine, limonene, amarogentine, humulone, lupulone, catechols, tan- nins), substances that prevent enzymatic browning (e.g. sulfite, ascorbic acid), ethereal oils, plant extracts, natural or synthetic colourings or colouring pigments (e.g. carotinoids, flavonoids, anthocyans, chlorophyll and derivatives thereof), spices, trigeminally active substances or plant extracts containing such trigeminally active substances, synthetic, natural or nature-identical flavorings or odorants as well as odour correctors. 25 [0042] Food compositions according to the invention, for example those in the form of preparations or semi-finished products, preferably comprise a flavor composition in order to complete and refine the taste and/or odour. A preparation can comprise as constituents a solid carrier and a flavor composition. Suitable flavor compositions comprise, for example, synthetic, natural or nature-identical flavorings, odorants and taste-imparting substances, reaction flavorings, smoke flavorings or other flavor-giving preparations (e.g. protein (partial) hydrolysates, preferably protein (partial) hydrolysates 30 having a high arginine content, barbecue flavorings, plant extracts, spices, spice preparations, vegetables and/or veg- etable preparations) as well as suitable auxiliary substances and carriers. Particularly suitable here are the flavor com- positions or constituents thereof which produce a roasted, meaty (in particular chicken, fish, seafood, beef, pork, lamb, mutton, goat), vegetable-like (in particular tomato, onion, garlic, celery, leek, mushroom, aubergine, seaweed), spicy (in particular black and white pepper, cardamom, nutmeg, pimento, mustard and mustard products), fried, yeast-like, boiled, 35 fatty, salty and/or pungent flavor impression and accordingly can enhance the spicy impression. The flavor compositions generally comprise more than one of the mentioned ingredients. [0043] The food compositions of the present invention are preferably selected from the group comprising
• confectionery, preferably reduced-calorie or calorie-free confectionery, preferably selected from the group compris- 40 ing muesli bar products, fruit gums, dragees, hard caramels and chewing gum,
• non-alcoholic drinks, preferably selected from the group comprising green tea, black tea, (green, black) tea drinks enriched with (green, black) tea extracts, rooibos tea, other herbal teas, fruit-containing low-sugar or sugar-free soft drinks, isotonic drinks, nectars, fruit and vegetable juices, fruit and vegetable juice preparations, 45 • instant drinks, preferably selected from the group comprising instant (green, black, rooibos, herbal) tea drinks,
• cereal products, preferably selected from the group comprising low-sugar and sugar-free breakfast cereals and muesli bars, 50 • dairy products, preferably selected from the group comprising reduced-fat and fat-free milk drinks, yoghurt, kefir, whey, buttermilk and ice-cream,
• products made from soy protein or other soybean fractions, preferably selected from the group comprising soy milk, 55 products produced from soy milk, drinks containing isolated or enzymatically treated soy protein, drinks containing soy flour, preparations containing soy lecithin, products produced from preparations containing soy lecithin and mixtures with fruit preparations and optionally flavors,
12 EP 3 501 296 A1
• sweetener preparations, tablets and sachets,
• sugar-free dragees,
5 • ice-cream, with or without milk-based constituents, preferably sugar-free.
FOOD ADDITIVES
[0044] The food compositions according to the present invention may further comprise components selected from the 10 group consisting of additional sweeteners or sweet-tasting compounds, aroma compounds, flavoring compounds and their mixtures.
Aroma or flavoring compounds
15 [0045] Aroma compounds and flavoring agents are well known in the art can be added to the flavor compositions of the invention. These flavoring agents can be chosen from synthetic flavoring liquid and/or oils derived from plants leaves, flowers,fruits and so forth,and combinations thereof. Representative flavoring liquids include:artificial, natural orsynthe tic fruit flavors such as eucalyptus, lemon, orange, banana, grape, lime, apricot and grapefruit oils and fruit essences including apple, strawberry, cherry, orange, pineapple and so forth; bean and nut derived flavors such as coffee, cocoa, 20 cola, peanut, almond and so forth; and root derived flavors such as licorice or ginger. [0046] The flavoring agent is preferably selected from the group consisting of essential oils and extracts, tinctures and balsams, such as, for example, anisole, basil oil, bergamot oil, bitter almond oil, camphor oil, citronella oil, lemon oil; Eucalyptus citriodora oil, eucalyptus oil, fennel oil, grapefruit oil, camomile oil, spearmint oil, caraway oil, lime oil, mand arin oil, nutmeg oil (in particular nutmeg blossom oil = maces oil, mace oil), myrrh oil, clove oil, clove blossom oil, orange oil, 25 oregano oil, parsley (seed) oil, peppermint oil, rosemary oil, sage oil (clary sage, Dalmatian or Spanish sage oil), star aniseed oil, thyme oil, vanilla extract, juniper oil (in particular juniper berry oil), wintergreen oil, cinnamon leaf oil; cin namon bark oil, and fractions thereof, or constituents isolated therefrom. [0047] It is of particular advantage if the flavored composition according to the invention comprises at least one flavoring agent, preferably two, three, four, five, six, seven, eight or more flavoring agents chosen from the following group: menthol 30 (preferably I-menthol and/or racemic menthol), anethole, anisole, anisaldehyde, anisyl alcohol, (racemic) neomenthol, eucalyptol (1,8-cineol), menthone (preferably L-menthone), isomenthone (preferably D-isomenthone), isopulegol, men- thyl acetate (preferably L-menthyl acetate), menthyl propionate, carvone (preferably (-)-carvone, optionally as a constit- uent of a spearmint oil), methyl salicylate (optionally as a constituent of a wintergreen oil), eugenol acetate, isoeugenol methyl ether, beta-homocyclocitral, eugenol, isobutyraldehyde, 3-octanol, dimethyl sulfide, hexanol, hexanal, trans-2- 35 hexenal, cis-3-hexenol, 4-terpineol, piperitone, linalool, 8-ocimenyl acetate, isoamyl alcohol, isovaleraldehyde, alpha- pinene, beta-pinene, limonene (preferably D-limonene, optionally as a constituent of an essential oil), piperitone, trans- sabinene hydrate, menthofuran, caryophyllene, germacrene D, cinnamaldehyde, mint lactone, thymol, gamma-octalac- tone, gamma-nonalactone, gamma-decalactone, (1,3E,5Z)-undecatriene, 2-butanone, ethyl formate, 3-octyl acetate, isoamyl isovalerate, cis- and trans-carvyl acetate, p-cymol, damascenone, damascone, cis-rose oxide, trans-rose oxide, 40 fenchol, acetaldehyde diethyl acetal, 1-ethoxyethyl acetate, cis-4-heptenal, cis-jasmone, methyl dihydrojasmonate, 2’- hydroxypropiophenone, menthyl methyl ether, myrtenyl acetate, 2-phenylethyl alcohol, 2-phenylethyl isobutyrate, 2- phenylethyl isovalerate, geraniol, nerol and viridiflorol. [0048] In particular preferred aroma or flavoring compounds encompass menthol, cineol, eugenol, thymol, cinnamic aldehyde, peppermint oil, spearmint oil, eucalyptus oil, thyme oil, cinnamon oil, clove oil, spruce needle oil, fennel oil, 45 sage oil, aniseed oil, star anise oil, chamomile oil, and caraway oil, and their mixtures.
Sweeteners and sweet-tasting substances
[0049] The term "sweeteners" here denotes substances having a relative sweetening power of at least 25, based on 50 the sweetening power of sucrose (which accordingly has a sweetening power of 1). Sweeteners to be used in an orally consumable product (in particular foodstuff, feed or medicament) according to the invention (a) are preferably non- cariogenic and/or have an energy content of not more than 5 kcal per gram of the orally consumable product. [0050] Advantageous sweeteners in a preferred orally consumable product (in particular foodstuff, feed or medicament) according to the invention are selected from the following groups (a1) and (a2): 55 [0051] Naturally occurring sweeteners, preferably selected from the group comprising
• miraculin, monellin, mabinlin, thaumatin, curculin, brazzein, pentaidin, D-phenylalanine, D-tryptophan, and extracts or fractions obtained from natural sources, comprising those amino acids and/or proteins, and the physiologically
13 EP 3 501 296 A1
acceptable salts of those amino acids and/or proteins, in particular the sodium, potassium, calcium or ammonium salts;
• neohesperidin dihydrochalcone, naringin dihydrochalcone, stevioside, steviolbioside, rebaudiosides, in particular 5 rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, dulcosides and rubusoside, suavioside A, suavioside B, suavioside G, suavioside H, suavioside I, suavioside J, baiyunoside 1, baiyunoside 2, phlomisoside 1, phlomisoside 2, phlomisoside 3 and phlomisoside 4, abrusoside A, abrusoside B, abrusoside C, abrusoside D, osladin, polypodoside A, strogin 1, strogin 2, strogin 4, selligueain A, dihydroquercetin 3-acetate, perillartin, telosmoside A 15, periandrin I-V, pterocaryosides, cyclocaryo- 10 sides, mukuroziocides, trans-anethole, trans-cinnamaldehyde, bryosides, bryonosides, bryonodulcosides, carnosi- flosides, scandenosides, trilobatin, phloridzin, dihydroflavanols, hematoxylin, cyanin, chlorogenic acid, albiziasa- ponin, telosmosides, gaudichaudioside, mogrosides, mogroside V, hernandulcins, monatin, phyllodulcin, glycyrrhe- tinic acid and derivatives thereof, in particular glycosides thereof such as glycyrrhizine, and the physiologically acceptable salts of those compounds, in particular the sodium, potassium, calcium or ammonium salts; 15 • extracts or concentrated fractions of the extracts, selected from the group comprising Thaumatococcus extracts (katamfe plant), extracts from Stevia rebaudiana, extracts from Momordica or Siratia grosvenorii (Luo-Han-Guo), extracts from Glycyrrhiza ssp. (in particular Glycyrrhiza glabra), extracts from Rubus ssp. (in particular Rubus suavis- simus), and extracts from Lippia dulcis; 20 [0052] Synthetic sweet-tasting substances, preferably selected from the group comprising magap, sodium cycla- mate or other physiologically acceptable salts of cyclamic acid, acesulfame K or other physiologically acceptable salts of acesulfame, neohesperidin dihydrochalcone, naringin dihydrochalcone, saccharin, saccharin sodium salt, aspartame, superaspartame, neotame, alitame, advantame, perillartin, sucralose, lugduname, carrelame, sucrononate and sucrooc- 25 tate. [0053] Suitable sweet-tasting substances, including natural sources of these substances such as for example
• sweet-tasting carbohydrates or sugars (e.g. sucrose (synonymous with saccharose), trehalose, lactose, maltose, melizitose, raffinose, palatinose, lactulose, D-fructose, D-glucose, D-galactose, L-rhamnose, D-sorbose, D-man- 30 nose, D-tagatose, D-arabinose, L-arabinose, D-ribose, D-glyceraldehyde, maltodextrin) or
• vegetable preparations containing predominantly these carbohydrates (e.g. from sugar beet (Beta vulgaris ssp., sugar fractions, sugar syrup, molasses), from sugar cane (Saccharum officinarum ssp., e.g. molasses, sugar syrups), from sugar maple (Acer ssp.), from agave (agave thick juice), 35 • synthetic/enzymatic hydrolysates of starch or sucrose (e.g. invert sugar syrup, highly enriched fructose syrups made from corn starch),
• fruit concentrates (e.g. from apples or pears, apple syrup, pear syrup), 40 • sugar alcohols (e.g. erythritol, threitol, arabitol, ribitol, xylitol, Sorbitol, mannitolol, dulcitol, lactitol),
• proteins (e.g. miraculin, monellin, thaumatin, curculin, brazzein),
45 • artificial sweeteners (magap, sodiumcyclamate, acesulfame K, neohesperidin dihydrochalcone, saccharin sodium salt, aspartame®, superaspartame, neotame, alitame, sucralose, lugduname, carrelame, sucrononate, sucrooctate, monatin),
• certain sweet-tasting amino acids (glycine, D-leucine, D-threonine, D-asparagine, D-phenylalanine, D-tryptophan, 50 L-proline),
• other sweet-tasting low-molecular substances, e.g. rebaudioside, stevioside, mogrosides, hernandulcin, phyllodul- cin, dihydrochalcone glycosides, glycyrrhizin, glycyrrhetinic acid ammonium salt or other glycyrrhetinic acid deriv- atives, 55 • extracts from sweet tasting plants, in particular Momordica grosvenori [Luo Han Guo] Hydrangea macrophylla, Stevia rebaudiana, Rubus suavissimus, Polypodium vulgare, Abrus precatorius, Pterocarya paliurus, Baccharis gaudichaudiana, Albizia myriophylla, Bryonia dioica, Phlomis betonicoides, Hemsleya carnosiflora, Lippia dulcis,
14 EP 3 501 296 A1
Glycyrrhiza sp. (liquorice), or individual sweet tasting substances isolated from those plants.
Thickeners
5 [0054] Advantageous thickeners in a preferred orally consumable product (in particular foodstuff, feed or medicament) according to the invention are selected from the group comprising: crosslinked polyacrylic acids and derivatives thereof, polysaccharides and derivatives thereof, such as xanthan gum, agar-agar, alginates or tyloses, cellulose derivatives, for example carboxymethylcellulose or hydroxycarboxymethylcellulose, fatty alcohols, monoglycerides and fatty acids, polyvinyl alcohol and polyvinylpyrrolidone. 10 [0055] Preference is given according to the invention to an orally consumable product (in particular foodstuff or feed) which comprises milk thickened with lactic acid bacteria and/or cream thickened with lactic acid bacteria and which preferably is selected from the group comprising yoghurt, kefir and quark. [0056] A food composition according to the invention comprising milk thickened with lactic acid bacteria and/or cream thickened with lactic acid bacteria is advantageously an orally consumable product which comprises a probiotic, wherein 15 the probiotic is preferably selected from the group comprising Bifidobacterium animalis subsp. lactis BB-12, Bifidobac- terium animalis subsp. lactis DN-173 010, Bifidobacterium animalis subsp. lactis HN019, Lactobacillus acidophilus LA5, Lactobacillus acidophilus NCFM, Lactobacillus johnsonii La1, Lactobacillus casei immunitass/defensis, Lactobacillus casei Shirota (DSM 20312), Lactobacillus casei CRL431, Lactobacillus reuteri (ATCC 55730) and Lactobacillus rham- nosus (ATCC 53013). 20 Additives for chewing gums
[0057] Particular preference is given to an orally consumable product (in particular foodstuff, feed or medicament) according to the invention that is a chewing gum and comprises a chewing-gum base. The chewing-gum base is preferably 25 selected from the group comprising chewing-gum or bubble-gum bases. The latter are softer, so that gum bubbles can also be formed therewith. Preferred chewing-gum bases according to the invention include, in addition to the natural resins or the natural latex chicle that are traditionally used, elastomers such as polyvinyl acetate (PVA), polyethylene, (low or medium molecular weight) polyisobutene (PIB), polybutadiene, isobutene-isoprene copolymers (butyl rubber), polyvinyethyl ether (PVE), polyvinylbutyl ether, copolymers of vinyl esters and vinyl ethers, styrene-butadiene copolymers 30 (styrene-butadiene rubber, SBR) or vinyl elastomers, for example based on vinyl acetate/vinyl laurate, vinyl acetate/vinyl stearate or ethylene/vinyl acetate, as well as mixtures of the mentioned elastomers, as described, for example, in EP 0 242 325, US 4,518,615, US 5,093,136, US 5,266,336, US 5,601,858 or US 6,986,709. In addition, chewing-gum bases that are preferably to be used according to the invention preferably comprise further constituents such as, for example, (mineral) fillers, plasticisers, emulsifiers, antioxidants, waxes, fats or fatty oils, such as, for example, hardened (hydro- 35 genated) vegetable or animal fats, mono-, di- or tri-glycerides. Suitable (mineral) illersf are, for example, calcium car- bonate, titanium dioxide, silicon dioxide, talcum, aluminium oxide, dicalcium phosphate, tricalcium phosphate, magne- sium hydroxide and mixtures thereof. Suitable plasticisers, or agents for preventing adhesion (detackifiers), are, for example, lanolin, stearic acid, sodium stearate, ethyl acetate, diacetin (glycerol diacetate), triacetin (glycerol triacetate), triethyl citrate. Suitable waxes are, for example, paraffin waxes, candelilla wax, carnauba wax, microcrystalline waxes 40 and polyethylene waxes. Suitable emulsifiers are, for example, phosphatides such as lecithin, mono- and di-glycerides of fatty acids, for example glycerol monostearate. [0058] Chewing gums according to the invention (in particular as disclosed above) preferably comprise constituents such as sugars of different types, sugar substitutes, other sweet-tasting substances, sugar alcohols (in particular Sorbitol, xylitol, mannitolol), ingredients having a cooling effect, taste correctors for unpleasant taste impressions, further taste- 45 modulating substances (e.g. inositol phosphate, nucleotides such as guanosine monophosphate, adenosine monophos- phate or other substances such as sodium glutamate or 2-phenoxypropionic acid), humectants, thickeners, emulsifiers, stabilisers, odour correctors and flavors (e.g. eucalyptus-menthol, cherry, strawberry, grapefruit, vanilla, banana, citrus, peach, blackcurrant, tropical fruits, ginger, coffee, cinnamon, combinations (of the mentioned flavors) with mint flavors as well as spearmint and peppermint on their own). The combination inter alia of the flavors with further substances that 50 have cooling, warming and/or mouth-watering properties is of particular interest.
Vitamins
[0059] Inanother embodimentof the presentinvention the compositions may include vitamins (component e1).Vitamins 55 have diverse biochemical functions. Some have hormone-like functions as regulators of mineral metabolism (e.g., vitamin D),or regulators ofcell andtissue growthand differentiation (e.g., some forms ofvitamin A). Others function as antioxidant s (e.g., vitamin E and sometimes vitamin C). The largest number of vitamins (e.g. B complex vitamins) act as precursors for enzyme cofactors, that help enzymes in their work as catalysts in metabolism. In this role, vitamins may be tightly
15 EP 3 501 296 A1
bound to enzymes as part of prosthetic groups: For example, biotin is part of enzymes involved in making fatty acids. Vitamins may also be less tightly bound to enzyme catalysts as coenzymes, detachable molecules that function to carry chemical groups or electrons between molecules. For example, folic acid carries various forms of carbon group - methyl, formyl, and methylene - in the cell. Although these roles in assisting enzyme-substrate reactions are vitamins’ best- 5 known function, the other vitamin functions are equally important. In the course of the present invention suitable vitamins are selected from the group consisting of
• Vitamin A (retinol, retinal, beta carotene),
10 • Vitamin B1 (thiamine),
• Vitamin B2 (riboflavin),
• Vitamin B3 (niacin, niacinamide), 15
• Vitamin B5 (panthothenic acid),
• Vitamin B6 (pyridoxine, pyridoxamine, paridoxal),
20 • Vitamin B7 (biotin),
• Vitamin B9 (folic acid, folinic acid),
• Vitamin B12 (cyanobalamin, hydoxycobalmin, methylcobalmin), 25 • Vitamin C (ascorbic acid),
• Vitamin D (cholecalciferol),
30 • Vitamin E (tocopherols, tocotrienols), and
• Vitamin K (phyolloquinone, menaquinone).
[0060] The preferred vitamins are ascorbic acid and tocopherols. Said vitamins may be present in the food composition 35 in amounts of about 0.1 to about 5 % b.w., and preferably about 0.5 to about 1 % b.w.
ORAL COMPOSITION
[0061] Another object of the present invention refers to an oral composition, comprising the sweetener composition 40 or the extract as disclosed above. These compositions may further contain additional sweeteners or sweet-tasting compounds, aroma compounds, flavoring compounds and their mixtures as already described above. [0062] Typical examples for non-food oral compositions encompass products for cleaning and protecting teeth and refreshing the oral cavity. [0063] The oral compositions of the present invention typically comprise an abrasive system (abrasive or polishing 45 agent), such as e.g. silicas, calcium carbonates, calcium phosphates, aluminium oxides and/or hydroxyapatites, surface- active substances, such as e.g. sodium lauryl sulfate, sodium lauryl sarcosinate and/or cocamidopropyl betaine, moisture- retaining agents, such as e.g. glycerol and/or Sorbitol, thickening agents, such as e.g. carboxymethylcellulose, polyeth- ylene glycols, carrageenan and/or Laponite®, sweeteners, such as e.g. saccharin, flavor correctants for unpleasant taste impressions, flavor correctants for further, as a rule not unpleasant taste impressions, flavor-modulating substances 50 (e.g. inositol phosphate, nucleotides, such as guanosine monophosphate, adenosine monophosphate or other sub- stances, such as sodium glutamate or 2-phenoxypropionic acid), cooling active compounds, such as e.g. menthol de- rivatives (e.g. L-menthyl lactate, L-menthyl alkyl carbonates, menthone ketals, menthanecarboxylic acid amides), 2,2,2- trialkylacetic acid amides (e.g. 2,2-diisopropylpropionic acid methylamide), icilin and icilin derivatives, stabilizers and active compounds, such as e.g. sodium fluoride, sodium monofluorophosphate, tin difluoride, quaternary ammonium 55 fluorides, zinc citrate, zinc sulfate, tin pyrophosphate, tin dichloride, mixtures of various pyrophosphates, triclosan, chlorhexidine, cetylpyridinium chloride, aluminium lactate, potassium citrate, potassium nitrate, potassium chloride, stron- tium chloride, hydrogen peroxide, aromas, sodium bicarbonate and/or odour correctants. [0064] Formulations or products according to the invention in the form of chewing gums or, in particular, dental care
16 EP 3 501 296 A1
chewing gums comprise chewing gum bases which comprise elastomers, such as, for example, polyvinyl acetates (PVA), polyethylenes, (low or medium molecular weight) polyisobutenes (PIB), polybutadienes, isobutene-isoprene copolymers (butyl rubber), polyvinyl ethyl ethers (PVE), polyvinyl butyl ethers, copolymers of vinyl esters and vinyl ethers, sty- rene/butadiene copolymers (styrene/butadiene rubber, SBR) or vinyl elastomers, e.g. based on vinyl acetate/ vinyl 5 laurate, vinyl acetate/vinyl stearate or ethylene/vinyl acetate, and mixtures of the elastomers mentioned, as described, for example, in EP 0 242 325, US 4,518,615, US 5,093,136, US 5,266,336 US 5,601,858 or US 6,986,709. In addition, chewing gum bases comprise further constituents, such as, for example, sugars, sugar substitutes or sweet-tasting substances in particular those described in WO 2009/21558, (mineral) fillers, plasticizers, emulsifiers, antioxidants, waxes, fats or fatty oils, such as, for example, hardened (hydrogenated) plant or animal fats, and mono-, di- or triglycerides. 10 Suitable (mineral) fillers are, for example, calcium carbonate, titanium dioxide, silicon dioxide, talc, aluminium oxide, dicalcium phosphate, tricalcium phosphate, magnesium hydroxide and mixtures thereof. Suitable plasticizers or agents for preventing sticking (detackifiers) are, for example, lanolin, stearic acid, sodium stearate, ethyl acetate, diacetin (glycerol diacetate), triacetin (glycerol triacetate) and triethyl citrate. Suitable waxes are, for example, paraffin waxes, candelilla wax, carnauba wax, microcrystalline waxes and polyethylene waxes. Suitable emulsifiers are, for example, 15 phosphatides, such as lecithin, and mono- and diglycerides of fatty acids, e.g. glycerol monostearate. [0065] Formulations or products according to the invention (in particular those which are in the form of an oral care formulation or product or in the form of a formulation) preferably additionally comprise one or more aroma and/or flavoring substances, such as essential oils and extracts, tinctures and balsams, such as, for example, anisole, basil oil, bergamot oil, bitter almond oil, camphor oil, citronella oil, lemon oil; Eucalyptus citriodora oil, eucalyptus oil, fennel oil, grapefruit 20 oil, camomile oil, spearmint oil, caraway oil, lime oil, mandarin oil, nutmeg oil (in particular nutmeg blossom oil = maces oil, mace oil), myrrh oil, clove oil, clove blossom oil, orange oil, oregano oil, parsley (seed) oil, peppermint oil, rosemary oil, sage oil (clary sage, Dalmatian or Spanish sage oil), star aniseed oil, thyme oil, vanilla extract, juniper oil (in partic ular juniper berry oil), wintergreen oil, cinnamon leaf oil; cinnamon bark oil, and fractions thereof, or constituents isolated therefrom. 25 [0066] It is of particular advantage if said formulations or products comprise at least one or more aroma substances, chosen from the following group: menthol (preferably I-menthol and/or racemic menthol), anethole, anisole, anisaldehyde, anisyl alcohol, (racemic) neomenthol, eucalyptol (1,8-cineol), menthone (preferably L-menthone), isomenthone (prefer- ably D-isomenthone), isopulegol, menthyl acetate (preferably L-menthyl acetate), menthyl propionate, carvone (prefer- ably (-)-carvone, optionally as a constituent of a spearmint oil), methyl salicylate (optionally as a constituent of a winter- 30 green oil), eugenol acetate, isoeugenol methyl ether, beta-homocyclocitral, eugenol, isobutyraldehyde, 3-octanol, dime- thyl sulfide, hexanol, hexanal, trans-2-hexenal, cis-3-hexenol, 4-terpineol, piperitone, linalool, 8-ocimenyl acetate, iso- amyl alcohol, isovaleraldehyde, alpha-pinene, beta-pinene, limonene (preferably D-limonene, optionally as a constituent of an essential oil), piperitone, trans-sabinene hydrate, menthofuran, caryophyllene, germacrene D, cinnamaldehyde, mint lactone, thymol, gamma-octalactone, gamma-nonalactone, gamma-decalactone, (1,3E,5Z)-undecatriene, 2-bu- 35 tanone, ethyl formate, 3-octyl acetate, isoamyl isovalerate, cis- and trans-carvyl acetate, p-cymol, damascenone, da- mascone, cis-rose oxide, trans-rose oxide, fenchol, acetaldehyde diethyl acetal, 1-ethoxyethyl acetate, cis-4-heptenal, cis-jasmone, methyl dihydrojasmonate, 2’-hydroxypropiophenone, menthyl methyl ether, myrtenyl acetate, 2-phenylethyl alcohol, 2-phenylethyl isobutyrate, 2-phenylethyl isovalerate, geraniol, nerol and viridiflorol.
40 PHARMACEUTICAL COMPOSITIONS
[0067] Another object of the present invention refers to a pharmaceutical composition, comprising the sweetener composition or the extract as disclosed above. These compositions may further contain additional sweeteners or sweet- tasting compounds, aroma compounds, flavoring compounds and their mixtures as already described above. 45 [0068] Pharmaceutical compositions according to the present invention may include similar additives as already ex- plained for the food and oral compositions, such as aroma and flavors. Pharmaceutical compositions may further include, oil bodies or emulsifiers and in particular co-actives supporting the beneficial properties of the pharmaceutical active agent. Therefore, the border between food compositions and pharmaceutical compositions is in flow and it should be understood that components cited for one application are recommended for the othermutatis-mutandis without literal 50 repetition.
INDUSTRIAL APPLICATION
[0069] Another object of the present invention refers to a method for creating or enhancing a sweeting effect in a food 55 or pharmaceutical composition encompassing adding an effective amount of the sweetening composition of Claim 1 or the extract of Claim 3 sufficient to produce the desired degree of sweetness to a composition that is intended for oral consumption. Usually, the sweetening composition or the extract are added to the food, the oral or the pharmaceutical composition in an amount of from 1 to about 2000 ppm - calculated on the final composition.
17 EP 3 501 296 A1
[0070] A final object of the present invention refers to the use of the sweetening composition or the extract as described above as a sweetener for food or pharmaceutical compositions. [0071] Preferably, one or more of the compound of formula (I) is used in an amount from 1 ppm to 2000 ppm by weight, based on the total weight of the composition and base on the total sum of all compounds of formula (I), if more than one 5 compound of formula (I) is used. More preferably, the sweetener compounds of formula (I) are used in an amount from 10 ppm to 1000 ppm by weight, most preferably in an amount of 20 ppm to 500 ppm by weight, based on the total weight of the composition and base on the total sum of all compounds of formula (I), if more than one compound of formula (I) is used.
10 EXAMPLES
[0072] The examples which follow are intended to illustrate the present invention without limiting the invention. Unless indicated otherwise all amounts, parts and percentages are based on the weight and the total amount or on the total weight and the total amount of the preparations. 15 EXAMPLE 1
Extraction and identification of compounds of formula (I)
20 Plant material
[0073] Leaves of Gynostemma pentaphyllum, were bought from Kuerzi special food import & distribution (Juval Kuerzi, Vicolo del mulino 32, 6648 Minusio, Switzerland) in November 2013. The plant material (batch GyTh2366) was collected in Thailand in 2013 (AnalytiCon Plant ID V-22072). 25 A) Extraction
[0074] 1.0 kg dried leaves of Gynostemma pentaphyllum were extracted with 12.51 methanol-MTB-ether at room temperature for 24h. The solvent was evaporated in vacuuo (yield: 62.8g extract, AnalytiCon extract-ID V-22072-W-01). 30 B) Pre-fractionation by reverse phase chromatography
[0075] The raw extract was subjected to a reverse phase medium pressure chromatography to enrich the saponins of interest. Reverse phase MPLC allows to remove sugars and lipids as well as other secondary metablolites like 35 flavoinoids very efficiently and to reach enriched fractions of saponins, which could be put on a second fractions step by HPLC using finer RP material. Sometimes it makes sense, to combine different MPLC prefractionations methods to get a more specific enrichment of selected saponins, e.g. by combination of RP 18 and RP4 material.
C-1911 40 [0076] 57.6 g raw extract V-22070-W-01 were separated by reverse phase medium pressure chromatography under the following conditions:
• stationary phase: RP-18, 40-63m (Merck) 45 • mobile phase solvent A: water
• mobile phase solvent B: methanol
50 • gradient 53% solvent A to 15% solvent A in 60 min
• column dimension: 50 x 250 mm
[0077] 8 Fractions were collected as set out in the following Table 1a: 55
18 EP 3 501 296 A1
Table 1a Fractions of C-1911 Fraction Volume [ml] yield [g] 5 C-1911-A 750 6,8550 C-1911-B 375 0,9074 C-1911-C 375 1,0124
10 C-1911-D 375 1,3762 C-1911-E 375 2,3550 C-1911-F 375 2,2650 HPLC C-1911-F C-1911-G 375 1,4962 15 C-1911-I 375 0,5849
C-2054
20 [0078] 62.8g raw extract V-22072-W-01 were separated by reverse phase medium pressure chromatography under the following conditions:
• stationary phase: RP-18, 40-63m (Merck)
25 • mobile phase solvent A: water
• mobile phase solvent B: methanol
• gradient 43% solvent A to 10% solvent A in 60 min 30 • column dimension: 50 x 250 mm
[0079] 16 Fractions were collected as set out in the following Table 1b:
35 Table 1b Fractions of C-2054 Fraction Volume [ml] yield [g] C-2054-Z-01 740 18,61 40 C-2054-Z-02 370 10,62 C-2054-Z-03 370 2,47 C-2054-Z-04 370 2,56
45 C-2054-Z-05 370 3,39 C-2054-Z-06 370 2,89 C-2054-Z-07 370 3,81 Z-07 and Z-08 were combined, further separation IDs C- 2054-E and C- 2054-R C-2054-Z-08 370 3,03 50 C-2054-Z-09 370 1,98 separation ID C-2054-F C-2054-Z-10 370 0,90 C-2054-Z-11 370 0,92
55 C-2054-Z-12 370 1,08 C-2054-Z-13 370 1,49 C-2054-Z-14 370 1,19
19 EP 3 501 296 A1
(continued)
Fractions of C-2054 Fraction Volume [ml] yield [g] 5 C-2054-Z-15 370 6,93 C-2054-Z-16 370 1,21
10 C-2562
[0080] 27.7g raw extract V-22072-W-01 were separated by reverse phase medium pressure chromatography under the following conditions:
m 15 • stationary phase: RP-18, 40-63 (Merck)
• mobile phase solvent A: water
• mobile phase solvent B: methanol
20 • gradient 43% solvent A to 15% solvent A in 60 min
• column dimension: 50 x 250 mm
25 [0081] 16 Fractions were initially collected and combined as set out in the following Table 1c:
Table 1c Fractions of C-2562 Fraction Volume [ml] yield [g] 30 C-2562-A 500 MPLC separation C-2624 C-2562-B to E 1610 5.24 HPLC separation C-2562-E and -U C-2562-F to C-2562-K 1850 12.64 MPLC separationC-2718
35 C-2718
[0082] 12.6g combined fractions form the MPLC C-2562 were separated by reverse phase medium pressure chro- matography using short chin RP material under the following conditions: 40 • stationary phase: RP-4, 40-63m (Merck)
• mobile phase solvent A: water
45 • mobile phase solvent B: methanol
• gradient 58% solvent A to 20% solvent A in 60 min
• column dimension: 50 x 250 mm 50 [0083] 10 Fractions were collected as set out in the following Table 1d:
Table 1d Fractions of C-2718 55 Fraction Volume [ml] yield [g] C-2718-A 500 0,0700
20 EP 3 501 296 A1
(continued)
Fractions of C-2718 Fraction Volume [ml] yield [g] 5 C-2718-B 500 0,0300 C-2718-C 370 0,2775 HPLC separation C-2718-C C-2718-D 370 2,8712 10 C-2718-E 370 2,3643 C-2718-F 370 2,1238 C-2718-G 370 1,6502 C-2718-H 370 0,9398 15 C-2718-I 370 0,4773 C-2718-K 370 0,1221
20 C) Second purification step by reverse phase chromatography
[0084] Pure compounds were isolated by reverse phase chromatography using different fractions from the first sep- aration step by MPLC. To reach a purity of >90% per compound sometimes several repeated separations by HPLC were necessary. Promising fractions from the preparative HPLC runs detected by light scattering detection ELSD were
25 analysed by LCMS. The solvent of all fractions containing compounds of interest was removed in vacuo followed by a freeze drying step. Isolated compounds were characterized by LCMS and 1D and 2D NMR spectroscopy. The following tables list preparative HPLC conditions used for the isolation of the compounds A to L.
Table 2a
30 Conditions of the separation of C-2201-B stationary phase Kromasil C18, 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B methanol + 5 mM ammoniumformate + 0.1 % formic acid 35 Flowrate 80 ml/min Gradient 48 - 59% B in 57 min Detection ELSD and UV 40 column dimension 50 x 250 mm used prefraction sample 0.78g of C-2201-B isolated compounds NP-021251: C-2201-B-01 (4.7 mg) NP-021252: C-2201-B-02 (15.5 mg) 45 NP-019871: C-2201-B-10 (98 mg)
Table 2b 50 Conditions of the separation of C-2054-E and C-2054-R stationary phase LichrospherSelect B, 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B acetonitrile/methanol = 1:1 + 5 mM ammoniumformate + 0.1 % formic acid 55 Flowrate 80 ml/min Gradient 47-69% B in 57 min
21 EP 3 501 296 A1
(continued)
Conditions of the separation of C-2054-E and C-2054-R Detection ELSD and UV 5 Column dimension 50 x 250 mm used prefraction sample 3.42 g of a combined fraction of C- 2054-Z-07 and Z-08
10 Table 2c Conditions of the separation of C-2054-F stationary phase LichrospherSelect B, 10 mm
15 mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B acetonitrile/methanol = 1:1 + 5 mM ammoniumformate + 0.1 % formic acid Flowrate 80 ml/min Gradient 50-70% B in 57 min 20 Detection ELSD and UV column dimension 50 x 250 mm used prefraction sample 1.98g of C-2054-F
25 Isolated compounds NP-019652: C-2054-F-06 (128 mg) NP-019654: C-2054-F-07 (318 mg)
[0085] Combination of HPLC fractions of C-2201 fractionation for further separation steps Based on LCMS and NMR analytics the following fractions were combined: 30
New separation ID Total amount [g] Combined fractions C-2201-A0.82 g C-2054-R-01, C-2054-D-03
35 C-2201-B0.78 g C-2054-R-02, C-2054-R-03, C-2054-F-02, C-2054-F-03 C-2201-CC-2054-F-04, C-2054-R-05 C-2201-DC-2054-E-02 C-2201-EC-2054-E-01 40
Table 2d Conditions of the separation of C-2201-A 45 stationary phase Kromasil C18, 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B methanol + 5 mM ammoniumformate + 0.1 % formic acid
50 Flowrate 80 ml/min Gradient 45 - 62% B in 57 min Detection ELSD and UV column dimension 50 x 250 mm 55 used prefraction sample 0.82g of C-2201-A Isolated compounds NP-021250: C-2049-A-06 (15 mg)
22 EP 3 501 296 A1
(continued)
Conditions of the separation of C-2201-A NP-019870: C-2049-A-10 (46 mg) 5
Table 2e Conditions of the separation of C-2201-B 10 stationary phase Kromasil C18, 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B methanol + 5 mM ammoniumformate + 0.1 % formic acid Flowrate 80 ml/min 15 Gradient 48 - 59% B in 57 min Detection ELSD and UV column dimension 50 x 250 mm 20 used prefraction sample 0.78g of C-2201-B isolated compounds NP-021251: C-2201-B-01 (4.7 mg) NP-021252: C-2201-B-02 (15.5 mg) NP-019871: C-2201-B-10 (98 mg) 25
Table 2f Conditions of the separation of C-2201-D 30 stationary phase Kromasil C18, 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B methanol + 5 mM ammoniumformate + 0.1 % formic acid Flowrate 80 ml/min 35 Gradient 48 - 59% B in 57 min Detection ELSD and UV column dimension 50 x 250 mm 40 used prefraction sample 0.47g of C-2201-D isolated compounds NP-021253: C-2201-D-02 (3.5 mg)
45 Table 2g Conditions of the separation of C-2718-C stationary phase Kromasil C18, 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid 50 mobile phase solvent B Acetonitril + 5 mM ammoniumformate + 0.1 % formic acid Flowrate 80 ml/min Gradient 14 - 34% B in 57 min 55 Detection ELSD and UV column dimension 50 x 250 mm
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(continued)
Conditions of the separation of C-2718-C stationary phase Kromasil C18, 10 mm 5 used prefraction sample 0.28g of C- 2718-C isolated compounds NP-019869: C-2718-C-07 (21.8 mg)
10 Table 2h Conditions of the separation of C-2626 stationary phase LichrospherSelect B. 10 mm
15 mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B acetonitrile/methanol = 1:1 + 5 mM ammoniumformate + 0.1 % formic acid Flowrate 80 ml/min Gradient 30-63% B in 57 min 20 Detection ELSD and UV column dimension 50 x 250 mm used prefraction sample 3x2.75g of combined fractions from different MPLCfractionations of the rawextract
25 Comment Corresponding fractions of 3 HPLC runs were combined Fraction collection Every 30 s (40 ml) Combined fractions Fraction Fraction # Yield [g] C-2626-A1-29 0.45 30 C-2626-B 30-36 0.43 C-2626-C 37-40 0.54 C-2626-D 41-46 0.33
35 C-2626-E 47-50 1.40 C-2626-F 51-55 3.14 C-2626-G 56-61 1.53 C-2626-H 62-64 0.46 40 C-2626-I 65-74 0.77 C-2626-K 75-85 0.31 C-2626-L 86-92 0.46 45 C-2626-M 93-101 0.56 C-2626-N 102-111 1.60 C-2626-O 112-120 0.27
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Table 2i Conditions of the separation of C-2726 stationary phase RP18. 40-63m 55 mobile phase solvent A water mobile phase solvent B Methanol
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Conditions of the separation of C-2726 Flowrate 80 ml/min 5 Gradient 72 - 10% Bin 57 min Detection ELSD and UV column dimension 50 x 250 mm 10 used prefraction sample 2.67g of C-2626-F Combined fractions Fraction Volume [ml] Yield [g] C-2726-A 500 0,20 C-2726-B 500 0,14 15 C-2726-C 370 0,37 C-2726-D 370 0,17 C-2726-E 370 0,28 20 C-2726-F 370 0,13 C-2726-G 370 0,16
25 Table 2j Conditions of the separation of C-2726-E stationary phase Kromasil C18, 10 mm mobile phase solvent A Water 30 mobile phase solvent B Acetonitril Flowrate 80 ml/min Gradient 25 - 35% B in 57 min
35 Detection ELSD and UV column dimension 50 x 250 mm used prefraction sample 0.28g of C- 2726-E Isolated compounds NP-022163: C-2726-E-01 (40 mg) 40 NP-022167: C-2726-E-03 (35 mg)
Table 2k 45 Conditions of the separation of C-1911-F stationary phase LichrospherSelect B. 10 mm mobile phase solvent A water + 5 mM ammoniumformate + 0.1 % formic acid mobile phase solvent B acetonitrile/methanol = 1:1 + 5 mM ammoniumformate + 0.1 % formic acid 50 Flowrate 80 ml/min Gradient 43 - 66% B in 57 min Detection ELSD and UV 55 column dimension 50 x 250 mm used prefraction sample 2.27g of C-1911-F
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Conditions of the separation of C-1911-F Isolated compounds NP-021249: C-1911-F-09 (41 mg) 5
D) Analytical characterization of isolated diterpene-glycosides
[0086] Fractions from preparative HPLC were collected (40ml each) and analyzed by HPLC-MS. Fractions containing 10 the same compound according to retention time and mass spectrum were combined, evaporated and analyzed by HPLC- MS and NMR ( 1H-NMR. HH-COSY. HSQC. HMBC). Structures were elucidated by interpretation of NMR and MS data.
Table 3 Conditions of the HPLC-MS of isolated compounds 15 HPLC HPLC PE series 200 MS System Applied Biosystems API 150 or API 165 datasystem Analyst 1.3
20 Stationary phase Phenomenex Luna C8 (2). 5 mm. 50 x 4.6 mm Flowrate 1.2 mL/min Detection (+/(-)-ESI. Fast-Switching-Mode . ELSD (Sedex 75) injection volume 10 mL 25 mobile phase: A: 5 mM Ammoniumformate and 0.1 % formic acid B: Acetonitrile/ Methanol= 1: 1 + 5 mM Ammoniumformate + 0.1 % formic acid (pH 3)
30 Gradient time [min] % A % B 0955 6 0 100
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E) Identification: analytical characterization of the compounds
[0087] The isolated compounds of formula (I) are characterized through mass spectroscopy and NMR spectroscopy, 40 see fig 1 to 24. The compounds were identified as:
Compound A (NP-019652)
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Compound B (NP-019654) 20 [0089]
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Compound D (NP-019870)
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Compound F (NP-022163)
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Compound G (NP-022167)
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Compound H (NP-021249)
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Compound I (NP-021250)
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25 Compound J (NP-021251)
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Compound L (NP-021253)
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EXAMPLE 2 50 Organoleptic test of the compounds of formula (I) against sucrose
[0100] The isolated pure compounds were dissolved in non-carbonated mineral water ("Evian") in a concentration of 0.4 mg/ml (400 ppm). The sweet taste of each sample was compared by a panel of 3-4 panelists with a solution of 55 sucrose in a concentration of 20 g/l.
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Compounds A to L were completely soluble in a concentration of 400 ppm.
[0101] The sweetness was evaluated as follows:
5 3 = sweeter than the control solution
2 = sweetness comparable with the control solution
1 = less sweet than the control solution but still sweet 10
Table 4 Organoleptic evaluation, test compound dissolved in water at 400 ppm
15 Compound compared to sucrose* Comment A 2 Slightly hot B 3 Slightly bitter C2 20 D3 E3 F3
25 G 2 Slightly bitter H 3 Slightly bitter I2 J2 30 K3 L2 * 2: isosweet to 2% Glucose solution; 3: sweeter than a 2% glucose so- lution 35
FORMULATION EXAMPLES
[0102] The following Tables 5a to 5d provide some examples for oral compositions comprising at least one of the 40 sweeteners disclosed before.
Table 5a Chewing gum, free of sugar; all amounts in % b.w.
45 Composition I II III Gum base 30.00 30.00 30.00 Sorbitol, powdered 40.00 40.00 40.00 Isomalt, powdered 9.50 9.50 9.50 50 Xylitol 2.00 2.00 2.00 Mannitol D 3.00 3.00 3.00 Compound A 0.10 - -
55 Compound B - 0.01 - Compound C - - 0.05 Emulgum / Plasticizing agent 0.30 0.30 0.30
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Chewing gum, free of sugar; all amounts in % b.w. Composition I II III 5 Sorbitol (70% water) 13.00 13.00 13.00 Spearmint aroma 1.00 1.00 1.00 Glycerol Ad 100
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Table 5b Tooth paste; all amounts in % b.w.
15 Composition IV V VI Glycerol 20.00 20.00 20.00 Solbrol M (sodium salt) 0.15 0.15 0.15 Sodium monofluor phosphate 0.76 0.76 0.76 20 Compound K 0.20 - - Compound L - 0.10 - Compound D - - 0.01
25 Dicalciumphosphate dihydrate 36.00 36.00 36.00 Aerosil 200 3.00 3.00 3.00 Sodium carboxymethyl cellulose 1.20 1.20 1.20 Sodium lauryl sulfate 1.30 1.30 1.30 30 Peppermint aroma 1.00 1.00 1.00 Deionised water Ad 100
35 Table 5c Mouth wash concentrate; all amounts in % b.w. Composition VII VIII IX
40 Ethanol 96% 42.00 42.00 42.00 Cremophor RH 455 5.00 5.00 5.00 Allantoin 0.20 0.20 0.20 Compound E 0.10 - - 45 Compound F - 0.01 - ColourL- Blue 5000 (1% in Wasser) 0.03 0.03 0.03 Spearmint aroma 2.00 2.00 2.00
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Table 5d
55 Hard boiled candy, sugar- free; all amounts in % b.w. Composition X XI XII Isomalt 94.98 94.98 94.98
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(continued)
Hard boiled candy, sugar- free; all amounts in % b.w. Composition X XI XII 5 Xylitol 2.40 2.40 2.40 Compound G 0.10 - - Compound H - 0.01 - 10 Citric acid 0.050 0.050 0.050 Cherry aroma 0.25 0.25 0.25 Water Ad 100
15 Table 5e Ice tea Composition XIII XIV XV 20 Sucrose 3.753.751.25 BlackTea Powder 0.25 0.25 0.25 Citric Acid 0.09 0.09 0.09
25 Potassium Sorbate 0.015 0.015 0.015 Peach Flavor 0.03 0.03 0.03 Compound I - 0.04 0.02 Compound J 0.05 0.04 - 30 Rebaudioside A - - 0.01 Water Ad 100
35 Table 5f Carbonated soft drink Composition XVI XVII XVIII
40 Sucrose 3.0 3.0 1.0 Cola Flavor 0.4 0.4 0.4 Phosphoric acid 85% 0.03 0.03 0.03 Caffeine 0.01 0.01 0.01 45 Compound C 0.05 0.02 Compound D 0.02 0.02 Acesulfam K 0.01
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[0103] The invention is further illustrated by analytical data provided in the following Figures 1 to 24:
Figure 1 Compound A 1H-NMR 55 Figure 2 Compound A LCMS Figure 3 Compound B 1H-NMR Figure 4 Compound B LCMS
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(continued) Figure 5 Compound C 1H-NMR Figure 6 Compound C LCMS 5 Figure 7 Compound D 1H-NMR Figure 8 Compound D LCMS Figure 9 Compound E 1H-NMR Figure 10 Compound E LCMS Figure 11 Compound F 1H-NMR 10 Figure 12 Compound F LCMS Figure 13 Compound G 1H-NMR Figure 14 Compound G LCMS Figure 15 Compound H 1H-NMR
15 Figure 16 Compound H LCMS Figure 17 Compound I 1H-NMR Figure 18 Compound I LCMS Figure 19 Compound J 1H-NMR Figure 20 Compound J LCMS 20 Figure 21 Compound K 1H-NMR Figure 22 Compound K LCMS Figure 23 Compound L 1H-NMR Figure 24 Compound L LCMS
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Claims
1. A sweetener composition comprising or consisting of at least one compound of general formula (I)
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wherein R1, R1a and R1b are independent from each other and represent OH, OGlucose, or OXylose, or OGalaktose, or OArabinose 55 R2 is one of the following structures
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with A1, A2 and A3 are independent from each other and represent H, OH, OOH, or OMe and B1 and B2 are independent from each other and represent H, OH, or O and the dotted line means a single or a double bond.
20 2. The composition of Claim 1, comprising or consisting of at least one compound selected from the group consisting of:
Compound A (NP-019652)
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Compound B (NP-019654)
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Compound C (NP-019869)
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Compound E (NP-019871)
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Compound G (NP-022167)
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Compound I (NP-021250)
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Compound J (NP-021251)
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Compound K (NP-021252)
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3. An extract comprising one or more of a compound of formula (I) obtainable by aqueous and/or alcoholic extraction of the plant Gynostemma pentaphyllum. 25 4. The extract of Claim 3, comprising at least one compound selected from the group consisting of:
Compound A (NP-019652)
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Compound C (NP-019869)
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Compound E (NP-019871)
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Compound G (NP-022167)
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5. A process for obtaining an extract of Gynostemma pentaphyllum rich in compounds according to formula (I) encom- passing or consisting of the following steps: 25
(a) Providing a suspension of roots, leaves or aerial parts ofGynostemma pentaphyllum in water, a C1-C4 aliphatic alcohol, acetone, ethyl acetate, methyl ethyl ketone, methyl- tbutyl ether, or their mixtures, (b) Subjecting said suspension to extraction using water, a 1C-C4 aliphatic alcohol, acetone, ethyl acetate, methyl ethyl ketone, methyl-tbutyl ether, or their mixtures at temperatures of about 20 to about 50 °C, and 30 optionally (c) Separating off the solvent.
6. A food composition, comprising the sweetener composition of Claim 1 or the extract of Claim 3.
35 7. The composition of Claim 6, further comprising components selected from the group consisting of additional sweet- eners or sweet-tasting compounds, aroma compounds, flavoring compounds and their mixtures.
8. An oral composition, comprising the sweetener composition of Claim 1 or the extract of Claim 3.
40 9. The composition of Claim 6, further comprising components selected from the group consisting of additional sweet- eners or sweet-tasting compounds, aroma compounds, flavoring compounds and their mixtures.
10. A pharmaceutical composition composition, comprising the sweetener composition of Claim 1 or the extract of Claim 3. 45 11. The composition of Claim 10, further comprising components selected from the group consisting of additional sweet- eners or sweet-tasting compounds, aroma compounds, flavoring compounds and their mixtures.
12. A method for creating or enhancing a sweetening effect in a food or pharmaceutical composition encompassing 50 adding an effective amount of the sweetening composition of Claim 1 or the extract of Claim 3 sufficient to produce the desired degree of sweetness to a composition that is intended for oral consumption.
13. The method of Claim 12, wherein the sweetening composition or the extract are added to the food, the oral or the pharmaceutical composition in an amount of from 1 to about 2000 ppm - calculated on the final composition. 55 14. The use of the sweetening composition of Claim 1 or the extract of Claim 3 as a sweetener for food or pharmaceutical compositions.
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15. The use according to Claim 14, wherein the sweetening composition or the extract are added to the food, the oral or the pharmaceutical composition in an amount of from 1 to about 2000 ppm - calculated on the final composition.
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REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
Patent documents cited in the description
• US 3087821 A, GENTILI [0003] • US 4518615 A [0057] [0064] • US 20120059071 A [0004] • US 5093136 A [0057] [0064] • EP 3099186 A2 [0004] • US 5266336 A [0057] [0064] • US 20110027413 A [0004] • US 5601858 A [0057] [0064] • EP 2529633 A1, SYMRISE [0006] • US 6986709 B [0057] [0064] • EP 0242325 A [0057] [0064] • WO 200921558 A [0064]
Non-patent literature cited in the description
• KASAI et al. AGRIC BIOL CHEM, 1989, 3347-3349 • KINGHORN et al. Terpenoid Glycoside Sweeteners [0004] in Natural Occurring Glycosides: Chemistry, Distribu- •MATSUMOTO. CHEM PHARM BULL, 1990, tion and Biological Properties. John Wiley and Sons, 2030-2032 [0004] 1999, 399-429 [0007] • LEMUS-MONDACA et al. FOOD CHEM, 2012, vol. • JUVAL KUERZI. Vicolo del mulino, November 2013, 132, 1121-1132 [0005] vol. 32, 6648 [0073]
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